CN110507655B

CN110507655B - Application of compound FG-4592 in preparation of pharmaceutical preparation for treating thyroid hormone receptor mediated diseases

Info

Publication number: CN110507655B
Application number: CN201810492095.1A
Authority: CN
Inventors: 李勇; 姚本强
Original assignee: Xiamen University
Current assignee: Xiamen University
Priority date: 2018-05-22
Filing date: 2018-05-22
Publication date: 2022-10-28
Anticipated expiration: 2038-05-22
Also published as: WO2019223688A1; CN110507655A

Abstract

The application of a compound FG-4592 in preparing a medicinal preparation for treating thyroid hormone receptor mediated diseases relates to the application of FG-4592 as a thyroid hormone receptor THR agonist. The compound FG-4592 or a pharmaceutically acceptable salt thereof can be applied to the preparation of an agonist of a thyroid hormone receptor, wherein THR comprises THR alpha and THR beta, and the agonist comprises a full agonist or a partial agonist. The compound FG-4592 or the pharmaceutically acceptable salt thereof can be applied to the preparation of a selective agonist of THR beta. FG-4592 is an agonist of the nuclear receptor THR β, and can regulate the function of THR β in metabolism such as energy metabolism, lipid metabolism, cholesterol-bile acid metabolism, inflammation, tumor, liver cirrhosis, blood progenitor cell differentiation, etc. involved in the body by binding to the target THR β.

Description

Application of compound FG-4592 in preparation of pharmaceutical preparation for treating thyroid hormone receptor mediated diseases

Technical Field

The invention relates to application of FG-4592 as a thyroid hormone receptor THR agonist, in particular to application of a compound FG-4592 in preparing a medicinal preparation for treating thyroid hormone receptor mediated diseases.

Background

The compound FG-4592, also called Roxadustat, has a CAS number of 808118-40-3 and a structural formula as follows:

FG-4592 is a HIF α prolyl hydroxylase inhibitor, and simultaneously induces EPO production, and is clinically useful in the treatment of anemia associated with chronic kidney disease and end stage renal disease. The oral small molecule stabilizes HIF1 alpha and HIF beta heterodimers by inhibiting hypoxia inducible factor HIF1 alpha hydroxylase, thereby regulating and controlling downstream target genes, promoting Erythropoietin (EPO) production, inhibiting inflammation, down-regulating hepcidin level (free iron-blocked hormone) and up-regulating other genes related to promoting iron, and ensuring effective mobilization and utilization of self-stored iron of human body. Erythropoiesis restoring the body's natural balance (Beuck, S., W.Schanzer, and M.Thevis, drug Testanal,2012.4 (11): p.830-45).

Nuclear receptors are ligand-activated transcription factors. Thyroid Hormone Receptors (THRs) are a very important member of the 48 nuclear receptors in humans, and there are two subtypes, THR α and THR β. The THRs promote various physiological activities of human body such as growth, differentiation and metabolism under the transcriptional activation regulation and control of natural ligand agonist thyroid hormones T3 and T4. The ligand-mediated pharmacological action principle of THR is that thyroid hormone ligand regulates and controls THR recruitment of various coactivators (or coactivators) to regulate transcription of downstream target genes by binding with the Ligand Binding Domain (LBD) of THR.

T4 and T3 produced by the thyroid are under multiple negative feedback regulation. TSH (also known as thyroid stimulating hormone), which is synthesized in the pituitary gland and whose secretion is controlled by thyroid stimulating hormone releasing hormone (TRH) synthesized in the hypothalamus, is responsible for normal thyroid function and thyroid hormone secretion regulation. At normal levels, this neuro, hormonal feedback regulation maintains the body weight, neometabolic rate, body temperature, mood and affects serum Low Density Lipoprotein (LDL) levels in humans. Therefore, there is weight gain, diabetes, hypertriglyceridemia, hypercholesterolemia, atherosclerosis, obesity, endocrine system diseases in the case of thyroid function deterioration or thyroid hormone resistance.

Hypothyroidism or hypothyroidism (hypothyroidism), abbreviated as hypothyroidism, refers to clinical syndrome caused by hypofunction of the body and various systemic functions due to deficiency of thyroid hormone caused by different reasons. The clinical manifestations include hypomnesis, mental retardation, ataxia; bradycardia, reduced cardiac output, and coronary heart disease; anorexia, abdominal distention, constipation, and pernicious anemia and iron deficiency anemia; flaccidity, pain, stiffness of muscles, and joint disorders such as chronic arthritis; female menorrhagia, amenorrhea due to chronic diseases, and infertility; impotence in men, hyposexuality; in severe cases, improper stress can induce coma, shock, heart and kidney failure, etc. Early mild cases were dominated by oral administration of thyroid tablets or agonists of Thyroid Hormone Receptor (THR) such as levothyroxine (national institutes of health) and authorized medical science popularization project dissemination network platform/encyclopedia medical network).

Thyroid hormone resistance Syndrome (SRTH) is also known as thyroid hormone intolerance or Thyroid Hormone Insensitivity Syndrome (THIS). There is a severe lack of therapeutic drugs for this syndrome. The disease can be divided into 3 types according to the onset and clinical manifestations: 1. generalized thyroid hormone atopy, which is characterized by the involvement of the pituitary and surrounding tissues, can be classified into thyroid function compensatory normal type and hypothyroidism type; 2. the selective pituitary gland is refractory to thyroid hormone, and is characterized in that the pituitary gland is affected mostly and does not react to thyroid hormone, while other peripheral tissues are not affected and can react normally to thyroid hormone, and hyperthyroidism appears in clinic; 3. the selective peripheral tissue is refractory to thyroid hormone, and the characteristic of the type is that the peripheral tissue is not responsive or insensitive to the thyroid hormone, but the pituitary is mostly unaffected and normally reacts to the thyroid hormone. The clinical manifestations are goiter, without deaf-mute and epiphyseal change, and although thyroid hormone and TSH are normal, the clinical manifestations are thyroid hypofunction, bradycardia, edema, hypodynamia, abdominal distension and constipation. This type can be manifested as poor intelligence, late development, and late bone maturation. Most thyroid hormone resistance syndromes are caused by the fact that the thyroid hormone receptor gene is mutated, so that the amino acid sequence of the thyroid hormone receptor is changed, the structure and the function of the receptor are changed, and the thyroid hormone T3 and T4 are resistant or insensitive. Therefore, the search for agonists of thyroid hormone receptors structurally distinct from the native thyroid hormones T3, T4 is expected to treat thyroid hormone resistance syndrome.

Hyperthyroidism is called hyperthyroidism for short, and is the disease that the hyperthyroidism is caused by hyperthyroidism and sympathetic nerve excitation due to the excessive release of thyroid hormone by thyroid synthesis, so as to cause palpitation, sweating, eating and frequent defecation and weight loss. Many patients also often have symptoms such as exophthalmos, eyelid edema, and visual deterioration.

THRs and cognitive disorders and depression:

affective disorders due to changes in thyroid hormone homeostasis are one of the causes of illness in individuals, although it is likely that patients will exhibit thyroid function normally for a short period of time. However, the incidence of clinically inadequate and excessive thyroid function appears to be a significantly increasing trend in a subset of patients. Thyroid hormones are essential for the development of the central nervous system, especially the brain. Mental patients are especially suffering from cognitive disorders and depression, and abnormalities in thyroid hormone metabolism and thyroid system immune function are crucial to the development of the disease. Thus thyroid hormones are frequently used to accelerate and enhance the treatment of antidepressant drugs, to support the maintenance of bipolar disorders for the treatment of affective disorders, even in patients with hypothyroidism (Bauer, m., et al., mol Psychiatry,2002.7 (2): p.140-56) where injection of thyroid hormone T4 to patients with hypothyroidism leads to an improvement in mood and learning ability, and positive experimental results are positively correlated with increased free thyroid hormone concentrations (bunevicus, r., currOpin Psychiatry,2009.22 (4): p.391-5).

Thyroid hormones have a major influence on the central nervous system, and psychiatric disorders, especially mood disorders, are often associated with disorders of thyroid hormone metabolism in the brain (Bauer, m., et al., JNeuroendocrinol,2008.20 (10): p.1101-14). T3 has long been used to treat patients with depression, either alone or in combination with other antidepressants, to enhance or accelerate the effects of antidepressant drugs on patients with thyroid depression. For example, T3 in combination with tricyclic antidepressants significantly accelerates tricyclic antidepressants in clinical trials for treating antidepressant patients, indicating significant efficacy of this treatment (Aronson, R., et al, arch Gen Psychiatry,1996.53 (9): p.842-8). In addition, the efficacy and safety of T3 in combination with another class of major antidepressant selective 5-hydroxytryptamine transporter inhibitors for the treatment of major depressive disorder has also been demonstrated (Cooper-Kazaz, R.and B.Lerer, int JNeuropsychopharmacol,2008.11 (5): p.685-99). These data indicate that specific enhancement of THRs activity is required to ensure normal development and maturation of the central nervous system, thereby enhancing metabolism, growth regulation and signaling in the nervous system, thereby increasing learning ability and avoiding cognitive disorders, and treating affective disorders such as depression.

THRs and high cholesterol series of diseases:

thyroid hormone has obvious effects of regulating lipid and cholesterol metabolism through THR beta, but the clinical application of natural thyroid hormone and thyroid receptor agonist to the treatment of hypercholesterolemia is only applied to patients with thyroid dysfunction. The reason is that non-specific agonists activate the THR α receptor, thereby causing negative effects in cardiovascular diseases. Therefore, the search for efficient and specific activation of THR β but not THR α will be a strong direction to treat a series of diseases caused by abnormal lipid metabolism and high cholesterol. Therefore, it is necessary to find effective and specific thyroid hormone receptor beta agonists, regulate lipid metabolism, and reduce cholesterol, thereby treating dyslipidemia, fatty liver, hypercholesterolemia, atherosclerosis and other diseases.

THRs and non-alcoholic steatitis:

recent clinical reports indicate that thyroid hormone receptor beta-specific agonists can protect liver tissue, promote liver function, and treat non-alcoholic fatty liver disease (NAFLD). The research and development of the method in the data published by Madrigal pharmaceutical company are praised as a brand new effective way for treating the nonalcoholic fatty hepatitis over FXR and PPAR alpha/delta. Independent of this finding, kim D et al, another scientist, found that subclinical hypothyroid patients had nonalcoholic steatohepatitis (NASH) and liver fibrosis that were directly related to their hypothyroidism. The proportion of nonalcoholic steatohepatitis and advanced fibrosis is higher in subjects with lower thyroid function. Patients with hypothyroidism have more severe hepatic steatosis, as well as more severe balloon and fibrosis (Kim, d., et al., clingasteroentolo hepatol,2018.16 (1): p.123-131e 1). Similarly, another group of scientists Miyake et al found that hyperthyroidism patients have a significant improvement in the pathological state of nonalcoholic steatohepatitis, and according to their research, the effect is related to the increase of liver enzyme level along with the increase of thyroid hormone level, and the liver enzyme level is increased along with the increase of thyroid hormone level. Therefore, hyperthyroidism can improve the pathological state of nonalcoholic steatohepatitis (Miyake, T., et al., intern Med,2016.55 (15): p.2019-23). These data show that prevention and treatment of non-alcoholic fatty liver disease, targeting of thyroid hormone receptor, and increasing receptor activity are effective means.

Thyroid hormone analogs development direction:

at present, in view of the effectiveness and universality of thyroid hormone receptors in treating diseases such as hypothyroidism, thyroid hormone resistance syndrome, depression and the like, the research and development of stronger thyroid hormone agonists is a main direction for overcoming. However, there are two major problems to be solved in this field of development, and first, as mentioned above, the native thyroid hormone T3 does not show any selectivity in binding to the two THR subtypes (THR α and THR β). Thus, administration of T3, while reducing plasma cholesterol, low Density Lipoprotein (LDL), triglyceride levels in animal models and humans, has limited its use due to the adverse effects of T3 on the heart, such as tachycardia, arrhythmia and muscle atrophy. Studies in knockout animals and the results of some selective ligands suggest that these cardiac side effects can be attributed to activation of THR α. Thus, existing thyroid hormone agonists have been developed primarily to increase selective activation of THR β and decrease activation of THR α. Another major problem is the adverse effects of gene mutations. Thyroid hormone resistance (Refetoff syndrome) describes a rare condition in which elevated levels of thyroid hormone are present, but Thyroid Stimulating Hormone (TSH) levels are not inhibited, or are not expected to be completely inhibited. Essentially, this reduces the thyroid hormone effector response to thyroid hormone (Refetoff, s., et al., J ClinEndocrino l meta, 1967.27 (2): p.279-94). Thus, despite the elevated serum levels of thyroid hormone, an individual may experience no change in thyroid function. The most common symptoms are goiter and tachycardia. It is also associated with some Attention Deficit Hyperactivity Disorders (ADHD) and depression (Hauser, P., et al., N Engl J Med,1993.328 (14): p.997-1001, fardella, C.E., et al., endocrine,2007.31 (3): p.272-8). The most common cause of this syndrome is mutation of the THR β gene, and more than 100 different thyroid hormone receptor β site mutations have been reported to cause thyroid hormone resistance syndrome, and new mutation sites have been reported (Beato-Vibora, p., j.et al., eur J obstet gynecol reprodiol, 2013.167 (1): p.118-9). The thyroid hormone resistance syndrome still lacks an effective treatment means at present, the mutation of thyroid hormone receptor protein enables original natural ligands such as T3 and a plurality of THR beta selective agonists to lose effects, and a brand-new medicine suitable for the polytype thyroid hormone resistance syndrome needs to be developed urgently.

Therefore, based on the above background, the development of more specific and selective thyroid hormone receptor ligands, especially THR β selective agonists and novel agonists that activate THRs mutants, has resulted in specific prevention and treatment of various diseases, such as hypothyroidism, thyroid hormone resistance syndrome, depression, etc., while drugs that avoid stimulation of the natural thyroid gland to the cardiovascular system and other toxicities are still highly urgent.

Disclosure of Invention

The invention aims to provide application of a compound FG-4592 (or a hydroxyquinine drug) in preparing an agonist of a Thyroid Hormone Receptor (THR) and application in preparing a medicinal preparation for preventing or treating diseases mediated by the thyroid hormone receptor.

The structural formula of the compound FG-4592 (or hydroxyl quinine drugs) is as follows:

the compound FG-4592 or a pharmaceutically acceptable salt thereof can be used for preparing an agonist of a Thyroid Hormone Receptor (THR), wherein the THR comprises THR alpha and THR beta, and the agonist comprises a full agonist or a partial agonist.

The compound FG-4592 or the pharmaceutically acceptable salt thereof can be applied to the preparation of a selective agonist of THR beta.

The compound FG-4592 or the pharmaceutically acceptable salt thereof can be applied to preparation of pharmaceutical preparations for preventing or treating thyroid hormone receptor-mediated diseases. The thyroid hormone receptor mediated diseases comprise hypothyroidism, polytype thyroid hormone resistance syndrome, mental diseases, nonalcoholic fatty liver disease and the like. Such psychiatric disorders include, but are not limited to, attention Deficit Hyperactivity Disorder (ADHD), depression, mental retardation, and cognitive dysfunction.

The polytype thyroid hormone resistance syndrome includes, but is not limited to, site lesions of mutations in the ligand binding domain of the clinically known THR beta receptor protein, including, but not limited to, the resulting thyroid hormone resistance syndrome V264D, A268 6242 zxft 62282S, V283A, M310T, E311K, S314C, A317T, R C, N3272 zxft 32332E, G3535 zxft 35346 3584 zxft 35346 4284 zxft 425384 zxft 5325 zxft 5623 zxft 45962 zxft 6262 459L, and the like.

The compound FG-4592 or the pharmaceutically acceptable salt thereof can be applied to the preparation of a pharmaceutical composition used in combination with other pharmaceutical preparations for preventing or treating thyroid hormone receptor mediated diseases.

The compound FG-4592 is a pharmaceutical composition comprising an effective dose of FG-4592 or a pharmaceutically acceptable salt thereof.

The compound FG-4592 has good THR (thyroid hormone) agonism effect, and experiments prove that the compound FG-4592 can be combined with a nuclear receptor Thyroid Hormone Receptor (THR) to induce the THR to recruit a coregulator and activate the expression of a target gene of the THR, so that the compound is a small molecular 'regulator' or 'THR ligand' of the nuclear receptor THR, and can be used as the THR agonist for preventing or treating THR-mediated related diseases. Wherein the THR agonist comprises a full or partial agonist of THR α and THR β.

AlphaScreen experiments demonstrated that FG-4592 induces the recruitment of co-activators by THR α and THR β. Since the coactivators were able to directly activate the THR downstream pathway, FG-4592 was shown to be an agonist of the nuclear receptor THR. Reporter gene experiments prove that FG-4592 can activate the transcriptional activity of nuclear receptors THR alpha and THR beta on target genes thereof, and indicate that FG-4592 is an agonist of the nuclear receptor THR. Crystallographic approaches further elucidate the binding mode of THR β binding to FG-4592 at the atomic level. Therefore, the agonists of FG-4592 thyroid hormone receptor THR can be proved to be used as thyroid hormone analogues to prevent or treat THR-mediated related diseases by using various methods.

Experiments prove that FG-4592 has obvious activation on the transcriptional activity of the THR beta mutant which generates the thyroid hormone resistance syndrome in a polytype way, and can be used for treating the thyroid hormone resistance syndrome. Since these mutants cause conformational changes in the THR Ligand Binding Domain (LBD), the strong binding capacity to thyroid hormones T3, T4 is lost. However, FG-4592 has a significantly different structure from thyroid hormones T3 and T4, so FG-4592 can bind to these THR beta receptor protein site pathogenic variants and significantly activate their transcriptional activity, so FG-4592 can be used to treat thyroid hormone resistance syndrome.

The experiments show that FG-4592 is an agonist of a nuclear receptor THR beta, and can regulate the functions of the THR beta in metabolism such as energy metabolism, lipid metabolism, cholesterol-cholic acid metabolism and the like, inflammation, tumor, liver cirrhosis, blood progenitor cell differentiation and the like which are involved in a human body by combining with a target THR beta.

FG-4592 is a hydroxyquinine drug capable of being orally taken as a small molecule for treating anemia of Chronic Kidney Disease (CKD), and is mainly used for a second generation hypoxia inducible factor, namely proline hydroxylase inhibitor (HIF-PHI) so as to treat anemia related to chronic kidney disease and end-stage kidney disease. So far, no medicine is available for treating thyroid hormone receptor mediated diseases such as thyroid hormone resistance, hypothyroidism or hypothyroidism and the like. Therefore, the FG-4592 provided by the invention has a treatment function in the aspects of diseases, and is a novel application method of FG-4592, which is inventive.

The thyroid hormone receptor mediated diseases such as thyroid hormone resistance disease, hypothyroidism or hypofunction and the like seriously affect the health and life of human beings, so the FG-4592 provided by the invention has a practical function in the aspect of preparing medicinal preparations for treating the serious diseases, has important social value and huge economic value and has practicability.

In some embodiments, the compounds of the methods of the invention may be formulated as pharmaceutical compositions for use in some dosing regimens. The pharmaceutical compositions of the invention may comprise the compound itself and a pharmaceutically acceptable salt or carrier therefor. Such compositions may also optionally comprise other therapeutic agents.

Certain agents or therapies may be administered in combination with the compounds of the present invention, such as matrix metalloproteinase inhibitors, lipoxygenase inhibitors, cytokine antagonists, immunosuppressive agents, cytokines, growth factors, immunomodulators, prostaglandins or anti-vascular hyperproliferative compounds.

The term "combination" and its related terms as used herein sequentially correspond to the administration of a therapeutic agent simultaneously or sequentially with a compound of the invention, e.g., the compound may be administered simultaneously or sequentially with another therapeutic agent in a single unit dosage form. Accordingly, the present invention provides a single unit dosage form comprising the compound, an additional therapeutic agent. In many dosing regimens, a patient or individual is generally considered to act in a "combination" if the patient or individual shows the relevant therapeutic effect of the agents used at the same time in a particular target tissue or sample (e.g., in the brain, in serum, etc.) when the patient or individual is exposed to at least two agents at the same time.

If these compounds are pharmaceutically acceptable salt preparations of the compounds of the present invention, the salts used should preferably be derived from inorganic or organic acids and bases. The acid salt includes: one of acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentane, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanes, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanic acid, tosylate, undecanoate, and the like. Alkali salts include ammonium salts, alkali metal salts, alkaline earth metal salts and organic alkali salts; the alkali metal salt includes sodium salt and potassium salt, the alkaline earth metal salt includes calcium salt and magnesium salt, and the organic base salt includes one of dicyclohexylamine salt, N-methyl-D-glucamine salt, or salt with amino acid such as arginine, lysine, etc.

The pharmaceutical compositions of the present invention may be formulated in solid or liquid form, including the following suitable administration forms: (1) Oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets, buccal, sublingual and systemic absorbents, boluses, powders, granules, pastes for sublingual use; (2) Parenteral administration, prepared as sterile solutions or suspensions or sustained release formulations by subcutaneous, intramuscular, intravenous or epidural injection; (3) Topical applications, such as creams, ointments, controlled release patches or sprays for the skin, lungs or oral cavity; (4) administration intrarectally, e.g., as an emulsion or foam; (5) others: sublingual, ophthalmic, transdermal or nasal, pulmonary and other mucosal ingestion.

Methods of preparing pharmaceutical compositions of the compounds include combining compound FG-4592 with a carrier or a plurality of accessory ingredients using any of the procedures described herein. In general, compound FG-4592 can be prepared in homogeneous intimate combination with a carrier (either a liquid carrier, a finely divided solid carrier, or both) and the product shaped as desired.

In some cases, prolonged efficacy can be achieved by slowing the rate of slow absorption of the drug from the subcutaneous or intramuscular injection, and this can be achieved by preparing liquid suspensions of crystalline or amorphous materials that are poorly water soluble. The rate of absorption of the drug depends on its rate of dissolution, which in turn may depend on crystal size and crystalline form. Alternatively, absorption by parenteral administration is delayed by dissolving or suspending the drug in an oily vehicle.

Injectable drug depot formulations can be prepared by combining the compound with a biodegradable polymer (e.g., polylactide-polyglycolide) in a microencapsulation matrix. Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of drug release can be controlled. Other biodegradable polymers include polyorthoesters and polyanhydrides. Injectable drug depot formulations compatible with body tissues can also be prepared by entrapping the drug in liposomes or microemulsions.

The pharmaceutical compositions of the present invention may be administered orally in any orally acceptable dosage form, including, but not limited to: capsules, tablets, and aqueous suspensions and solutions. For oral tablets, lactose and corn starch are usually coated, usually with a lubricant, such as magnesium stearate. For oral capsule formulations, useful diluents include lactose and dried corn starch. When administered orally in aqueous suspensions and solutions, as well as propylene glycol formulations, the pharmaceutically active ingredients are combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.

The pharmaceutical composition of the present invention can be administered by making it into an aerosol or an inhalant. Such agents may be prepared according to techniques widely used in the art of pharmaceutical formulation, and may also be prepared as saline solutions. Such prior art often uses benzyl alcohol or other suitable preservatives, fluorocarbons and/or other solubilizing or dispersing agents, absorption promoters to enhance their bioavailability.

An additional advantage of transdermal patches is the controlled delivery of the compounds of the present invention to the body. Such dosage forms may be made by dissolving or dispersing the compound in a suitable medium. Absorption enhancers may be used to increase the absorption of the compound by the skin. Control of the rate of passage of the compound through the skin can also be achieved by employing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

The invention obtains FG-4592 as a specific ligand of THR through high-throughput screening. The effect of compounds inducing THR with co-activators or co-inhibitors was examined by AlphaScreen biochemical methods.

Based on luciferase reporter gene activity analysis and molecular structure level demonstration in cell transfection experiments, specific selective recognition between the receptor and the ligand is further verified, and the transcriptional activation effect of the compound on THR is further verified.

And (3) selecting polytype THR beta receptor protein site disease variants generating thyroid hormone resistance syndrome, wherein the thyroid hormone T3 loses significant activation on the transcription of reporter genes of all the mutants in the table, but FG-4592 has significant activation on the transcriptional activity of the THR beta mutants.

THR beta/FG-4592 is prepared into a compound, and the compound is crystallized, subjected to X-ray crystal diffraction and structurally resolved through crystallography, so that the binding mode of THR beta and FG-4592 which are mutually bound is clarified from the atomic level. Therefore, by combining various methods such as biochemistry, molecular biology and structural biology, FG-4592 is discovered and verified to be a novel THR agonist. Like thyroid hormone T3, FG-4592 can activate THR-mediated transcriptional activity and can also remarkably activate THR beta mutant transcriptional activity which is resistant to thyroid hormone T3.

Drawings

FIG. 1 shows that FG-4592 promotes the interaction of the SRC co-activator LXXLL motif with THR α and THR β.

FIG. 2 is a three-dimensional crystal structure diagram of a complex in which FG-4592 is bonded to THR β. The band diagram represents the structure of the nuclear receptor THR β protein, labeled with the name of the relevant alpha helix. The bar shows the structure of the ligand FG-4592 compound.

FIG. 3 is a two-dimensional schematic of the amino acid interaction in the ligand binding domain of FG-4592 with the thyroid hormone receptor THR β. The hydrophobic alkyl end of the ligand forms hydrophobic interaction with nearby nonpolar amino acid, while the carboxyl end of the ligand mainly directly forms hydrogen bond with polar amino acid or forms hydrogen bond network with polar amino acid through water. Wherein the grey arrows and black lines indicate the formation of hydrogen bonds and hydrophobic interactions of the ligands with the amino acid residues, respectively.

Detailed Description

Example 1, FG-4592 was shown to enhance the recruitment of coactivators of THR α and THR β, and is an agonist of the thyroid hormone receptor THR.

FIG. 1 shows that FG-4592 promotes the interaction of the SRC co-activator LXXLL motif with THR α and THR β. Using the AlphaScreen technique, FG-4592 at a final concentration of 1 μ M was tested for its effect on the interaction of LBD of THR α with Η R β with a variety of co-regulatory factors including within SRCLXXLL motifs.

The N-terminal biotinylated cofactor polypeptide sequence was:

SRC1-2：SPSSHSSLTERHKILHRLLQEGSP；

SRC2-3：QEPVSPKKKENALLRYLLDKDDTKD；

SRC3-3：PDAASKHKQLSELLRGGSG；

NCOR-1：GQVPRTHRLITLADHICQIITQDFARNQ；

NCOR-2：GHSFADPASNLGLEDIIRKALMGSF。

protein purification: the human THR α LBD (amino acid residue numbers 148-410) and THR β LBD (amino acid residue numbers 202-461) genes were constructed in pET24a vector containing a 6 polyhistidine fusion tag, manufactured by Novagen, inc., respectively. The constructed vector was transformed into E.coli BL21 (DE 3) and cultured at 30 ℃ to OD600 of about 0.8, then cooled to 22 ℃ and 0.1mM isopropyl 1-thio-. Beta. -D-galactoside (IPTG) was added to induce expression of the target protein for 6h. After subsequent centrifugation (4200 r.p.m.) for 30min, the E.coli cells were resuspended in buffer (25mM Tris PH7.5,25mM imidazole, 150m sodium chloride) on ice and frozen at-70 ℃ for 2h before cell disruption by sonication. The lysed cell fluid was centrifuged (> 20000r.p.m.) at 4 ℃ for 30min, and the supernatant was applied to a GE nickel ion exchange column (NiSO 4-loaded HiTrap HP column, GE Healthcare). Subsequently, the sample-loaded nickel column was eluted in AKTApure from GE using a gradient of 25 to 500mM imidazole in elution buffer. The eluted protein was further purified using an anion exchange column (Q-Sepharose column).

In the process of searching for THR ligands, the LBD proteins of THR α and THR β were used as "baits" to screen compound libraries using AlphaScreen technology. Polypeptides of various motifs required for binding of THR LBD protein to ligand can be detected by an AlphaScreen kit (Perkins-Elmer) experiment (Jin et al, nature communications 2013,4,1937). The reaction system for this experiment was 20-80nM of the receptor LBD protein, 20nM of biotinylated cofactor polypeptide, 5. Mu.g/ml of donor and acceptor glass beads, buffer (25mMHepes, 100mMNaCl and 0.1mg/ml of bovine serum album, pH 7.0). This technology has been widely used in drug development based on the interaction of nuclear receptors with ligands.

Half maximal effect concentration (EC 50) at which FG-4592 induces binding of THRs to LXXLL motifs as shown in table 1, FG-4592 greatly enhanced recruitment of THR α and THR β to coactivators SRC1, SRC2 and SRC3, but had no effect on the recruitment of cosuppressors, relative to control DMSO (dimethyl sulfoxide), indicating that FG-4592 is an agonist of THR α and THR β. Moreover, the effect of FG-4592 on inducing THR beta to recruit polytype coactivators is obviously stronger than that of THR alpha.

TABLE 1

Further detection with a concentration gradient FG-4592alphaScreen showed that FG-4592 induces a significantly lower half maximal effect concentration (EC 50) for THR β binding to the LXXLL motif than THR α, and thus FG-4592 has a more selective activation of THR β.

Example 2, FG-4592 was shown to enhance transcriptional activation of the THR reporter gene and to be an agonist of the thyroid hormone receptor THR.

To further give the activation effect of FG-4592 on THR, a plasmid encoding Gal4DNA binding domain, reporter gene, THRLBD was co-transfected into Cos7 cells. Cos-7 cells were cultured in DMEM medium containing 10% fetal bovine serum, and transient transfection was performed using Lipofectamine 2000 (Invitrogen) kit. In the Gal-4 driven reporter gene experiments, 200ng of Gal4-LBD was co-transfected with 200ng of pG5Luc (Promega). Agonist compounds were added 5h after transfection. After 24h of treatment, cells were harvested for luciferase assay experiments. Fluorescence detection experiments were performed by co-transfection of Renilla reporter genes for endogenous reference. The experimental result is consistent with the AlphaScreen detection result, FG-4592 can obviously enhance the transcriptional activation of the reporter genes of THR alpha and THR beta, the efficacy (efficacy) of the compound (5 mu M) for respectively inducing the transcriptional activity of the wild type and mutant reporter genes of THR alpha and THR beta is shown in the table 2, and the fact that the agonist of FG-4592 thyroid hormone receptor THR can activate the THR mediated gene expression at the cellular level is shown again.

TABLE 2

Example 3 demonstrates that FG-4592 has a significant activation of the transcriptional activity of the THR β mutant and can be used to treat thyroid hormone resistance syndrome.

Table 2 lists The THR beta receptor protein site disease variants selected for The polytype to produce thyroid hormone resistance syndrome (The HumanGene Mutation Database, http:// www.hgmd.cf.ac.uk). Since these mutants cause conformational changes in the THR Ligand Binding Domain (LBD), the strong binding capacity to thyroid hormones T3, T4 is lost. THR β plasmid mutagenesis was performed using the Quick-Change site-directed mutagenesis kit (Stratagene) and the Gal-4 driven reporter gene experiment was performed as in example 2. As shown in table 2, significant activation of thyroid hormone T3 transcription of the reporter genes was lost for all mutants in the table. However, because FG-4592 has a significantly different structure from thyroid hormones T3 and T4, FG-4592 can bind to these THR beta receptor protein site pathogenic variants and significantly activate their transcriptional activity, and thus FG-4592 can be used for treating thyroid hormone resistance syndrome.

Example 4 demonstrates a more selective activation of THR β by FG-4592.

As described above, thyroid Hormone Receptors (THRs) have two subtypes, THR α and THR β, and activation of THR α has a certain side effect on the cardiovascular system and the like. Thus, the native thyroid hormone T3 does not show any selectivity in binding to the two THR subtypes (THR α and THR β), and existing thyroid hormone agonists were developed primarily to increase the selective activation of THR β and decrease the activation of THR α. A Gal-4 driven reporter gene experiment as in example 2 was performed for THR α and THR β to obtain compound activation potency (efficacy), and potency EC was calculated using the full dose curve ₅₀ The potency ratio and potency EC50 ratio of compounds to induce transcriptional activity of THR β and THR α reporter genes, respectively, are shown in table 3. Potency is a term of pharmacology of a drug and refers to the ability of a drug to produce the greatest effect, and is used to evaluate the magnitude of the greatest effect of different drugs. Potency (potency): also called potency, refers to the dose or concentration of a drug required to produce a certain effect. EC (EC) ₅₀ Is the half effect concentration, i.e. the dose of drug that causes 50% of the subjects to produce a particular effect. The smaller the EC50, the better the activity and the stronger the strengthIs large. As shown in table 3, FG-4592 is more selectively activated for THR β in both potency and potency than T3, and is thus a THR β selective agonist.

TABLE 3

Example 5 demonstrates the crystal structure resolution of the complex in which FG-4592 binds to THR β.

In order to reveal the molecular mechanism of FG-4592 and THR β recognizing and binding to each other from the atomic level, the crystal structure of FG-4592 and THR β forming a complex was resolved conventionally (fig. 2). The THR β protein was obtained as in example 1, and FG4592 in an amount 5 times the amount of the purified THR β LBD protein and the coactivator SRC2-3 polypeptide (ENALLRYLLDKD) in an amount 2 times the amount of the THR β LBD protein were added, and after incubating on ice for 1 hour, the mixture was concentrated to 10mg/mL. During crystallization, a crystallization screening kit of Hampton company is used, and a suspension drop method is used for crystallization screening after a sample compound is mixed with a screening buffer solution at a ratio of 1: 1. The crystal grows best in about 48h at room temperature, with the best conditions: 0.2M sodium citrate, 20% by volume polyethylene glycol 3350, and directly freezing and storing the crystal in liquid nitrogen after adding a cryoprotectant. The frozen crystals are collected by the synchrotron radiation center of Shanghai countries, and CCP4 tool software is used for determining the structure after the data is processed and reduced by HKL 3000. And manually modifying the structure by using REFMAC and the like after the Coot software is modified, and completing structure analysis after the structure parameter evaluation is completed.

The structure shows that FG-4592 binds to the THR β ligand binding domain in a classical "sandwich" conformation. From the three-dimensional structure diagram, FG-4592 is clearly present in the ligand binding pocket of THR β (fig. 2). In the complex structure, the THR β AF-2 helix bound to FG-4592 forms a "pocket" together with helices H3, H4 and H5, which interacts with the LXXLL motif of the co-activator SRC 2. This is a typical pattern of interaction of nuclear receptors with agonists. Detailed structure-activity relationship analysis showed that FG-4592 forms a series of hydrophobic, hydrogen-bonding and van der waals interactions with amino acids in the ligand binding domain of thyroid hormone receptor THR β (fig. 3), which regulate the binding of FG-4592 to thyroid hormone receptor THR β. These structural analyses strongly demonstrate the typical mechanism by which FG-4592 regulates activation of the thyroid hormone receptor THR.

The invention relates to a hydroxyquinine compound, and provides application of the hydroxyquinine compound in preparing an agonist of a Thyroid Hormone Receptor (THR) and preparing a pharmaceutical preparation for preventing or treating thyroid hormone receptor-mediated diseases. The present invention relates to the modulation of thyroid hormone receptor activity, particularly thyroid hormone receptor beta (THR β) activity, for use in the prevention or treatment of thyroid hormone receptor mediated diseases such as hypothyroidism, thyroid hormone resistance and depression.

Sequence listing

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Claims

1. The application of an agonist FG-4592 of a thyroid hormone receptor or a pharmaceutically acceptable salt thereof in preparing a medicament for preventing or treating diseases mediated by the thyroid hormone receptor, wherein the thyroid hormone receptor is shown as THR, and the structural formula of the compound FG-4592 is as follows:

the thyroid hormone receptor mediated diseases comprise hypothyroidism, hypothyroidism and multiple thyroid hormone resistance syndrome.

2. The use of claim 1, wherein THR comprises THR α and THR β.

3. The use of claim 1, wherein said agonist comprises a full agonist or a partial agonist.

4. Use according to claim 2, wherein THR β is a selective agonist of THR β.

5. The use as claimed in claim 1 wherein said polytype of thyroid hormone resistance syndrome includes, but is not limited to, site lesions of mutations in the ligand binding domain of the clinically known THR β receptor protein, said site mutations including, but not limited to, the resulting thyroid hormone resistance syndrome V264D, A268D, R282S, V283A, M310T, E311K, S C, A317T, R C, N3272 zxft 32332E, G3535 zxft 3584 zxft 35346 4284 zxft 5353535325 zxft 5323 zxft 5623 459 6262 zxft 62459L.

6. The use of the agonist of thyroid hormone receptor FG-4592 or a pharmaceutically acceptable salt thereof according to claim 1 for the preparation of a medicament for the prevention or treatment of thyroid hormone receptor mediated diseases, wherein said medicament is used in combination with other pharmaceutical preparations for the prevention or treatment of thyroid hormone receptor mediated diseases.